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Growth and Reproduction Assay
Day -1 (solutions can be made earlier) *prepare S-media (add the following to a 100mL bottle of S-basal) ** 1mL Potassium Citrate ** 1mL Trace Metals Solution ** 300uL Calcium Chloride ** 300uL MgSO4 *Bleach 8-10 NGM plates full of gravid N2s and grow overnight in S-media to stage L1s *Also grow 2 bottles of OP50 in LB Day 0 *Concentrate 200mL OP50 culture **spin at 4000rpm for 5 minutes in 50ml conical tubes in tabletop centrifuge **pour off supernatant and resuspend in 20ml S-media for 10xOP50 **dilute 1:5 for 2xOP50 and again 1:5 for 0.4xOP50 *Add 2ml of appropriate OP50 concentration to wells in a 12 well tissue culture plate *Store remaining OP50 at RT, but best to make fresh each day *add approximately 2000 L1 N2 worms to each well **spin L1s at 2000rpm for 2 minutes, remove supernatant, resuspend in 5mL fresh S-media **spot 4x 2uL onto slide and count; calculate # L1s/uL **add appropriate volume to each flask *incubate in plastic box on orbital shaker tray (100-120 rpm) at 20°C *Photograph 20-50 leftover L1s **place 20-50 individuals on unseeded NGM plate **spread out individuals for better images **photograph with dissecting scope for day 0 measurements **also photograph a 1mm scale bar each time the zoom is changed **rename files to avoid forbidden characters **convert to 8-bit grayscale .tif files (in Fiji: Image -> Type -> 8-bit) **Measure with WormSizer (remember to pass/fail as needed) **Convert: average mass (nanograms) = 1.08 * average volume (picoliters) Day 1 *Wash a sample of 20-50 worms 2-3x with M9 in a 1.7ml Eppendorf tube to remove OP50 **spinning at 3600rpm for 30 seconds (or in smaller benchtop centrifuge briefly) *Photograph as above for day 1 measurements Day 2-end *Photograph as above *Count 2-hour progeny production **prepare 10+ unseeded NGM plates per group by adding 100uL appropriate OP50 **place 10+ individuals from 12-well plate onto NGM plate **pick single individuals into droplets of liquid **replenish appropriate OP50 as needed, about every 30 minutes **after 2 hours, pick parents off plate **incubate plates for 2-3 days, then count progeny Filter daily after progeny are present (suggestion: separate protocol for filtration) *Place a clean 30 micron nylon filter into filter holder (and then autoclave them together?) *pass some M9 through filter to moisten it *draw up a population into a clean 3ml (or 6ml) syringe; attach to filter holder *place filter over 15 ml conical tube, and slowly pass the worms through the filter *use a few ml of M9 to rinse worms from the well and syringe into the filter holder *pass additional M9 through filter to wash worms through *connect tubing to outlet *place filter holder upside down over a new 15 ml conical tube *pass M9 through to release larger worms from filter *centrifuge the product to concentrate adult worms *check for presence of L1s and other small worms in the “adult” fraction *check yield and look for trapped adult worms on filter *if backflow is insufficient, may need to wash adult worms off filter by vortexing the filter in a 50ml conical tube half-full of M9 Category:Protocols Category:Population Dynamics